Cells have numerous strategies to cope with the consequences of stresses that cause protein misfolding and aggregation, leading to formation of plaques, fibrils, and other aggregated species encountered in aging cells, cataract, and neurodegenerative diseases. The protein chaperones known as small heat shock proteins are the cell's first responders and are therefore key to maintenance of cellular health. Despite their name, human small heat shock proteins (sHSP) are rarely called up to respond to the stress of elevated temperature, as humans have effective and tight regulation of temperature. Nevertheless, much of the current knowledge of sHSPs has been obtained under elevated temperature conditions where they are highly active. In this project, we seek to understand the molecular mechanisms by which human sHSPs respond to physiologically relevant stresses and how they maintain cellular proteins in soluble forms. Of relevance to the National Eye Institute, the sHSPs to be focused on are highly expressed in ocular tissues (lens, retina, cornea) as well as in other tissues. Dysfunction and aberrant expression of HSPB1 and HSPB5 are associated with ocular diseases including cataract, diabetic retinopathy, and age-related macular degeneration that together account for 65% of blindness worldwide. Perhaps linked to their particular functional niche in which they must respond to small changes in cellular conditions such as pH and metal ion concentrations, human sHSPs have evolved properties that are unique in the protein world. They exist as large (> 100 kDa) polydisperse and dynamic assemblies that defy conventional structural biology approaches. We will apply emerging technologies capable of providing molecular and possible atomic-level information about heterogeneous and dynamic systems. Structural information obtained from solution- and solid-state NMR, negative-stain and cryo-EM, and native mass spectrometry will be integrated with dynamic information from FRET-based subunit dynamics and NMR and with functional information from activity assays and client-binding experiments to define the mechanism(s) by which HSPB5 and HSPB1 are activated by conditions brought about by stresses relevant to tissues of the eye such as hypoxia, ischemia, and UV exposure. Both wild-type sHSPs and forms carrying inherited disease mutations will be investigated.